A sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis

Abstract

The predominant testicular gap junctional protein connexin43 (cx43) is located between neighboring Sertoli cells (SCs) and between SCs and germ cells. It is assumed to be involved in testicular development, cell differentiation, initiation, and maintenance of spermatogenesis with alterations of its expression being correlated with various testicular disorders. Because total disruption of the cx43 gene leads to perinatal death, we generated a conditional cx43 knockout (KO) mouse using the Cre/loxP recombination system, which lacks the cx43 gene solely in SCs (SCCx43KO), to evaluate the SC-specific functions of cx43 on spermatogenesis in vivo. Adult SCCx43KO(-/-) mice showed normal testis descent and development of the urogenital tract, but testis size and weight were drastically lower compared with heterozygous and wild-type littermates. Histological analysis and quantitation of mRNA expression of germ cell-specific marker genes revealed a significant reduction in the number of spermatogonia but increased SC numbers/tubule with only a few tubules left showing normal spermatogenesis. Thus, SC-specific deletion of cx43 mostly resulted in an arrest of spermatogenesis at the level of spermatogonia or SC-only syndrome and in intratubular SC clusters. Our data demonstrate for the first time that cx43 expression in SCs is an absolute requirement for normal testicular development and spermatogenesis.

Figure 1

PCR genotyping with primers UMP and UMPR (Cx43 flox PCR) and Cre26 and MT1 (Cre PCR) using DNA isolated from mouse tails of representative SCCx43KO^−/− (A), SCCx43KO^+/− (B), and WT males (C) of the offspring (F2 generation). The single 1.1-kb fragment (white arrow) indicates homozygosity for cx43flox (A, lane 2) and the Cre band (black arrowhead) at 1.914 bp (A, lane 4) confirms Cre expression. The double bands at 1.1 kb (white arrow) and 0.987 kb (black arrow) indicate heterozygosity for cx43flox (B, lane 2), and the Cre band (black arrowhead) at 1.914 bp (B, lane 4) confirms Cre expression. PCR genotyping of a WT male shows only a single band at 987 bp (black arrow), indicating that no floxed allele(s) (C, lane 2) are present, and no band for Cre (C, lane 4), confirming its absence. Lane 1 (A–C), 100-bp DNA ladder. Lane 3 (A–C), 0.5-kb DNA ladder.

Figure 2

Dissection of urogenital tract of WT (A) and genital tracts of SCCx43KO^+/− (B) and SCCx43KO^−/− (C) mice at the age of 170 days. On dissection, the testes of SCCx43KO^−/− were found to be located in exactly the same location as in heterozygous littermates (testes are marked with a dotted line, insets B and C). Note that the size of the testes in SCCx43KO^−/− mice was drastically reduced compared with testes of SCCx43KO^+/− and WT littermates. t, testis; e, epididymidis; dd, ductus deferens; sv, seminal vesicles. Scale bars = 1 cm.

Figure 3

Comparison of testis weights in adult SCCx43KO^−/− mice. Total testis weights were significantly reduced (P < 0.001) in adult SCCx43KO^−/− mice (range, 60 to 120 days) compared with WT and SCCx43KO^+/− controls. Data are shown as mean ± SEM (n = 9 per genotype).

Figure 4

Comparison between testes obtained from adult WT, SCCx43KO^+/−, and SCCx43KO^−/− mice. H&E staining of testes from day 170 WT (A), SCCx43KO^+/− (B), and SCCx43KO^−/− mice (C and G) revealed clear differences in germ cell content. Complete spermatogenesis was observed in WT (A) and heterozygous males (B), but smaller tubules with SCO syndrome (not shown) or spermatogenic arrest at the level of spermatogonia (C and G) or residual spermatogenesis (G, right tubule) occurred in SCCx43KO^−/− animals. Corresponding sections of cauda epididymidis showed that sperm could only be detected in WT (D) and SCCx43KO^+/− (E), but not in SCCx43KO^−/− males (F). Lc, Leydig cells; black arrowheads, SC nuclei; black arrows, spermatogonia; transparent arrows, primary spermatocytes; white arrowheads, round spermatids; transparent arrowheads, elongated spermatids. Scale bars = 50 μm.

Figure 5

Comparison of germ cell (spermatogonia) counts (from H&E staining) at days 30, 60, 90, and 120 postpartum in WT, SCCx43KO^+/−, and SCCx43KO^−/− mice. Twenty circular tubules were examined for each germ cell count, genotype, and postpubertal age. Given are means ± SD over seminiferous tubules within each animal. Significance between genotypes, P < 0.0001; between ages, n.s.

Figure 6

Comparison of SC counts (from H&E staining) at days 30, 60, 90, and 120 postpartum in WT, SCCx43KO^+/−, and SCCx43KO^−/− mice. Twenty circular tubules were examined for each SC count, genotype, and postpubertal age. Given are means ± SD over seminiferous tubules within each animal. Significance between genotypes, P < 0.001; between ages, n.s.

Figure 7

Specificity and efficiency of the deletion of the Cx43 gene in SCs. PCR analysis of cx43 deletion in testis (lane 2), heart (lane 3), and tail (lane 4) of SCCx43KO^−/− mice and testis (lane 5) of WT mice. Only testicular samples from SCCx43KO^−/− males show a band at 670 bp (black arrowhead), indicating deletion of the cx43 floxed allele. Lane 1, 0.5-kb DNA ladder.

Figure 8

Comparison between the testes obtained from adult WT (A, D, and G), SCCx43KO^+/− (B, E, and H), and SCCx43KO^−/− (C, F, and I) mice. Results of β-gal immunostaining (A–C), cx43 in situ hybridization (D–F), and cx43 immunostaining (G–I). As expected, SC nuclei of WT mice (A, black arrowheads) revealed no β-gal immunoreactivity. In seminiferous tubules of SCCx43KO^+/− (B) and SCCx43KO^−/− (C) mice, Cre-mediated deletion of one or both alleles of the cx43 gene could only be detected in SCs by nuclear β-gal immunostaining but not in germ cells or interstitial Leydig cells. Note the few β-gal-immunonegative spermatogonia (C, black arrows). In situ hybridization of seminiferous tubules from WT animals (D) and SCCx43KO^+/− (E) showed that cx43 mRNA was mainly localized around the nuclei of SCs, spermatogonia, and spermatocytes. Only a weak signal was detectable in the cytoplasm of round spermatids. In contrast, only few spermatogonia were found to express cx43 mRNA in seminiferous tubules of SCCx43KO^−/− mice (F), whereas SCs displayed no signal of cx43 gene expression. In the seminiferous epithelium of WT (G) and SCCx43KO^+/− males (H), cx43 is found to be immunolocalized between SCs and between SCs and spermatogonia/primary spermatocytes. In contrast, no immunostaining at all was detected in seminiferous tubules from SCCx43KO^−/− mice (I), indicating that neither SCs nor spermatogonia are able to synthesize cx43 protein. Note the single spermatogonium (I, black arrow) and intratubular SC cluster (I, transparent arrow). Heart tissue from SCCx43KO^−/− mice revealed cx43-immunopositive intercalated disks (I, inset). Scale bars = 50 μm.

Figure 9

Representative RT-PCR for prm1 (A), prm2 (B), and tnp1 (C) using testes homogenates from SCCx43KO^−/− males (A–C, lane 2), SCCx43KO^+/− males (A–C, lane 3), and WT mice (A–C, lane 4). Specific mRNA for prm1 and prm2 (black arrows) was only detectable in SCCx43KO^+/− and WT mice (A and B, lanes 3 and 4), indicating the presence of spermatids but the absence in SCCx43KO^−/− (A and B, lane 2). Interestingly, a weak band for tnp1 (black arrow) could be detected in SCCx43KO^−/− animals (C, lane 2) compared with a strong expression in SCCx43KO^+/− mice (C, lane 3) and WT males (C, lane 4). Lane 1 (A–C), 100-bp DNA ladder.